SKF96365 modulates activity of CatSper channels in human sperm

Abstract Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+ concentration ([Ca2+]i) transient followed by repetitive [Ca2+]i activity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P = 0.00004). In non-pre-treated cells, SKF had an effect similar to P4, inducing a [Ca2+]i transient in >80% of cells which was followed by oscillations in ≈50% of cells. The CatSper blocker RU1968 (11 µM) inhibited the SKF-induced [Ca2+]i increase and reversibly arrested [Ca2+]i oscillations. Using whole-cell patch clamp, we observed that SKF enhanced CatSper currents by 100% within 30 s, but amplitude then decayed to levels below control over the next minute. When cells were stimulated with P4, CatSper currents were stably increased (by 200%). Application of SKF then returned current amplitude to control level or less. When sperm were prepared in medium lacking bovine serum albumin (BSA), both P4 and SKF induced a [Ca2+]i transient in >95% of cells but the ability of SKF to induce oscillations was greatly reduced (P = 0.0009). We conclude that SKF, similar to a range of small organic molecules, activates CatSper channels, but that a secondary blocking action also occurs, which was detected only during patch-clamp recording. The failure of SKF to induce oscillations when cells were prepared without BSA emphasizes that the drug does not fully mimic the actions of P4.


Introduction
Intracellular Ca 2þ concentration ([Ca 2þ ] i ) signalling is crucial to sperm function, being a key regulator of motility, acrosome reaction, and capacitation (Darszon et al., 2005;Publicover et al., 2008;Prajapati et al., 2022). A number of stimuli, including zona pellucida (ZP), are believed to regulate human sperm function by this pathway, the best characterized being the steroid progesterone (P4), which is secreted by the cells of the cumulus and is present throughout the female tract (Correia et al., 2007). P4 acts on human sperm by activation of the sperm-specific Ca 2þ -permeable channel CatSper (Lishko et al., 2011;Strunker et al., 2011), which, in human sperm and those of many other species, is the principal mechanism of Ca 2þ mobilization (Lishko et al., 2012;Lishko and Mannowetz, 2018).
In addition to CatSper, Ca 2þ stores and capacitative (storeoperated) Ca 2þ entry (CCE) are implicated in sperm [Ca 2þ ] i signalling (Costello et al., 2009;Correia et al., 2015). CCE may be triggered by CatSper-mediated elevation of [Ca 2þ ] i , serving to amplify, propagate and/or prolong the [Ca 2þ ] i signal (Alasmari et al., 2013;Correia et al., 2015). No specific pharmacological agents are available for blockade of CCE, but 10-30 mM SKF96365 (SKF) is a known and well-characterized antagonist of CCE both in cell lines and in a range of acutely cultured cell types (Labelle et al., 2007;Ng et al., 2008Ng et al., , 2010Li et al., 2010;Sabourin et al., 2016). SKF has also been shown to inhibit calcium-release activated Ca 2þ (CRAC) current (a form of CCE; Parekh and Putney, 2005) in cells where CRAC currents occur naturally and also in cells transfected with Orai, a pore forming channel which is involved in CRAC and other CCE channels (Prakriya and Lewis, 2002;Yeromin et al., 2006;Varnai et al., 2009). SKF has been used as a tool to investigate the participation of CCE in sperm functions. Treatment with SKF inhibited acrosome reaction in sea urchin sperm (Hirohashi and Vacquier, 2003) and suppressed the increase in [Ca 2þ ] i induced by mitochondrial inhibitors (Ardon et al., 2009). In mouse sperm, SKF inhibited Ca 2þ influx and acrosome reaction induced by maitotoxin (Trevino et al., 2006) and suppressed motility (Ru et al., 2015). In human sperm, SKF has been reported to suppress motility (Castellano et al., 2003) to inhibit acrosomal swelling (believed to be a key stage in acrosome reaction) induced by P4 and by thapsigargin, which mobilizes stored Ca 2þ ; Sosa et al., 2016), and to inhibit acrosome reaction induced by recombinant ZP3 (Jose et al., 2010).
Human sperm stimulated with P4 generate an 'instantaneous' CatSper-mediated Ca 2þ transient, which is followed by a plateau of increased [Ca 2þ ] i upon which [Ca 2þ ] i oscillations are often superimposed (Harper et al., 2004;Kirkman-Brown et al., 2004;Aitken and McLaughlin, 2007;Sanchez-Cardenas et al., 2014;Mata-Martinez et al., 2018). Pharmacological manipulations suggest that this latter part of the [Ca 2þ ] i response may involve secondary release of stored Ca 2þ (Harper et al., 2004;Servin-Vences et al., 2012;Correia et al., 2015;Mata-Martinez et al., 2018), andCCE (Lefiè vre et al., 2012;Mata-Martinez et al., 2018). We therefore investigated the effect of SKF on both P4-induced and basal [Ca 2þ ] i signalling in human sperm. SKF might be expected to suppress [Ca 2þ ] i signalling in human sperm. Instead, we observed a dose-dependent increase in [Ca 2þ ] i upon SKF-treatment. This effect of SKF resembled the well-characterized action of P4 on human sperm and further investigation confirmed that the drug modulates activity of CatSper channels.

Materials
All chemicals were obtained from Sigma-Aldrich (Poole, UK) except fluo4-AM (acetoxymethylester), which was from Thermo Fisher Scientific, Swindon, UK. As the observed effect of SKF was unexpected, we tested the drug from four different suppliers [Calbiochem/Merck (UK), Tocris (Abingdon, UK), Abcam (Cambridge, UK), and Sigma-Aldrich]. Similar results were obtained in every case. Fluo4-AM was prepared in dimethylsulphoxide (DMSO) containing 20% Pluronic F-127 (Thermo Fisher). P4 and RU1968 were dissolved in DMSO at 10 mM and diluted in supplemented Earle's balanced salt solution (sEBBS) prior to use. The concentration of DMSO in imaging experiments was 0.03-0.3%. RU1968 was a kind gift of Dr Timo Strü nker, Centre of Reproductive Medicine and Andrology, Mü nster, Germany.

Selection and preparation of spermatozoa
Written consent was obtained from donors in accordance with the Human Fertilisation and Embryology Authority (HFEA) Code of Practice (versions 7 and 8) under local ethical approval (South Birmingham Local Ethical Committee (Ref: 2003/239), and University of Birmingham (ERN 07-009 and ERN-12-0570)). Samples were obtained by masturbation after 2-3 days sexual abstinence. After liquefaction (30 min), sperm were swum up into sEBSS (60 min), adjusted to a maximum of %6Â10 6 /ml and left to capacitate (36 C, 5.5% CO 2 ) for 5 h.

Collection and analysis of imaging data
Imaging was carried out as described (Nash et al., 2010). Briefly, after adjusting sperm concentration to 1.5Â10 6 /ml, the cell suspension was divided into aliquots of 200 ll and incubated with fluo4-AM (5 lM) for 30 min (36 C, 5.5% CO 2 ). Cells were then transferred to a perfusable imaging chamber, the base of which was a coverslip coated with 0.001% poly-D-lysine and incubated for an additional 5 min to allow cells to settle. The chamber was installed on the stage of an inverted fluorescence microscope (Nikon TE300) and perfused with sEBSS to remove unattached cells and excess dye. All experiments were performed at 25 C in a continuous flow of sEBSS, with a perfusion rate of 0.6 ml/min. Fluorescence excitation was at 470-480 nm (Zeiss or Cairn 75 W xenon source with filter or OptoLED, Cairn, UK) and emission at 520 nm. Images were captured at 0.1-0.2 Hz using a 40Â or 60Â oil-immersion objective and a Hamamatsu Orca 1 or Q Imaging Rolera-XR CCD camera or an Andor Ixon 897 EMCCD camera controlled by iQ software (Andor Technology, Belfast, UK). Stimuli were applied to the cells by inclusion in the perfusing medium.
Analysis of images and background correction was done using iQ software (Andor Technology). Regions of interest were drawn around the posterior head/neck of each cell and the background subtracted. Average intensity was obtained for each area, imported into Microsoft Excel and normalized by calculating the percentage change in fluorescence (DF) using the equation: where DF is the percentage change in fluorescence intensity at time t, F is fluorescence intensity at time t and Frest is the mean of !10 determinations of F during the control period before application of any stimulus. For comparison of the amplitudes of P4 and SKF-induced [Ca 2þ ] i signals, a mean response of all cells was calculated for each experiment. SKF and P4-induced transient amplitudes were calculated as the increment in DF (the difference between the DF values at the signal peak and immediately before onset of the signal) and statistical tests were carried out using these values, unless stated otherwise.

Electrophysiology
Capacitated cells were re-suspended in standard bath solution and allowed to adhere to glass coverslips that were transferred to an inverted microscope where they were superfused with standard bath solution Whole cell CatSper currents were then recorded using Cs þ -based divalent-free pipette and bath solutions (Lishko et al., 2011). Currents were evoked by a ramp protocol (À80 to 80 mV over 1 s), which was repeated at 2 s intervals to allow continuous monitoring of current amplitude. Membrane potential was held at 0 mV between ramps. Data were sampled at 2 kHz, filtered at 1 kHz.

Statistics
Data were assessed for normality using the Anderson-Darling test and tested accordingly. Percentage data were transformed using the arcsine square root conversion (Sokal and Rohlf, 1981) before statistical analysis to allow application of parametric tests. Chi-square test was used for categorical variables (with adjustment for multiple testing as appropriate). The Student's t-test (paired or independent), Mann-Whitney or Wilcoxon test, with adjustment for multiple testing as appropriate, were used for continuous variables. Data are presented as mean6SEM. Statistical analysis was carried out using Minitab 18 (Minitab, PA, USA).  Supplementary  Fig. S1), and also significantly increased the proportion of cells in which [Ca 2þ ] i oscillations were observed (81.9 6 3.8%; P ¼ 0.00004; Fig. 1c). We therefore investigated the effects of SKF without prior exposure to P4.

SKF increases [Ca 21 ] i in human sperm
Upon application of 30 mM SKF, 87 6 3% of cells generated an immediate [Ca 2þ ] i transient (1-2 min duration) or slowly developing plateau ( Fig. 2a and b; n ¼ 9 experiments, P ¼ 0.01 compared to 3 mM P4), followed by oscillations in 56 6 7% of cells ( Fig. 2a and b, P ¼ 0.7 compared to 3 mM P4). The initial [Ca 2þ ] i response evoked by SKF was clearly smaller than that seen with P4 in parallel experiments (P ¼ 0.00006; Fig. 2c) and also than the [Ca 2þ ] i transients observed when SKF when was applied following pretreatment with P4 (P ¼ 0.003; compare responses to SKF in Figs 1b and 2c). When cells were exposed to P4 after pre-treatment with SKF, we observed a [Ca 2þ ] i transient of similar amplitude to that induced by P4 under control conditions (P ¼ 0.78; Fig. 2a and d).
To provide a better comparison of efficacy of the two stimuli, we used population fluorimetry to study the dose-dependence of [Ca 2þ ] i transient amplitudes induced by SKF and P4 ( Fig. 3a and  b). The EC 50 values obtained by fitting curves to the data for SKF (n ¼ 6 experiments) and for P4 (n ¼ 7 experiments) were 9.5 and 0.032 mM, respectively. Furthermore, the saturating amplitude of the SKF-induced response was <30% of that for P4 (Fig. 3b). Vehicle (DMSO) at the doses used had negligible effect (Achikanu et al., 2018). stimulated cells with SKF to induce [Ca 2þ ] i oscillations, then applied 11 mM RU1968. Similarly to the effect of the drug on P4-induced oscillations, the proportion of cells in which oscillations occurred was reduced from 73.2 6 10.4% to 5.5 6 1.5% (P ¼ 0.017; n ¼ 3 experiments) but oscillations recovered upon washout of the CatSper blocker and persisted until washout of SKF (Fig. 4b). Since CatSper is sensitive to intracellular alkalinization, we checked whether SKF modified intracellular pH. We observed no effect of SKF, whereas 4-aminopyridine (a weak base that rapidly increases pHi in human sperm; Alasmari et al., 2013) increased pHi by almost 0.6 units (Fig. 4c). These findings suggest that SKF can stimulate CatSper, via a pH-independent mechanism, as has been described previously for a number of small organic compounds (Brenker et al., 2012;Tavares et al., 2013;Schiffer et al., 2014;Birch et al., 2022). We therefore used patch clamp directly to measure the effect of SKF treatment on monovalent (Cs þ ) CatSper currents. Consistent with the effects of SKF on [Ca 2þ ] i , CatSper currents increased 'immediately' upon application of SKF, reaching a maximum after 20-30 s (P ¼ 0.024; n ¼ 6 cells; Fig. 5a and b). However, current amplitude then decreased over the next 30-60 s, falling to values below pre-stimulus levels (P ¼ 0.01; Fig. 5a and b). When cells were exposed to P4 (3 mM), inward and outward currents rapidly increased, reaching a plateau within 30 s (P < 0.01; n ¼ 3 cells; Fig. 5c and d). Subsequent application of SKF caused a brief increase in current amplitude (more clearly visible in inward than outward currents), but currents then decreased over the next 40-50 s, reaching levels at or below those recorded before stimulation ( Fig. 5c and d).

Capacitation and the action of SKF
To further compare the abilities of P4 and SKF to induce oscillations, we investigated the dependence of oscillations on prior incubation of the cells in capacitating medium. We therefore carried out a series of experiments using a modified medium from which BSA was omitted, a procedure which impairs cytoplasmic alkalinization during capacitation of human sperm (Zhao et al., 2021). As reported previously (Bedu-Addo et al., 2005), this procedure did not abolish the P4-induced [Ca 2þ ] i transient (present in 99% of cells), but significantly reduced its amplitude from 155 6 13% under standard conditions (Fig. 1b) to 96 6 10% ( Fig. 6a and b; P ¼ 0.025). [Ca 2þ ] i oscillations occurred in 56 6 5% of cells ( Fig. 6a and d). However, in cells stimulated with SKF, >95% responded to the drug with an initial [Ca 2þ ] i transient, which was similar in amplitude to that seen with P4 (number 6 SD; Fig. 6b and c), but only 24.3 6 6.8 generated any repetitive activity (typically 3 cycles, e.g. Fig. 6c, red trace) and just 2.3 6 1.4% generated sustained, repetitive activity similar to that seen with P4 ( Fig. 6d; P ¼ 0.0009 compared to P4). Instead, the [Ca 2þ ] i transient was typically followed by a slowly rising plateau (Fig. 6c). CatSper is known to be highly promiscuous, being activated by a wide variety of small organic molecules including a range of pharmacological tools, insecticides, and sunscreens (Brenker et al., 2012;Tavares et al., 2013;Schiffer et al., 2014;Birch et al., 2022). It is also possible that the SKF-induced ] i activity (red bar shows SKF application) by exposure to 11 mM RU1968 in the superfusing saline (grey bar). When RU1968 was washed out, oscillations recovered and continued until SKF was removed. (c) fluorimetric assessment of pHi in a population of human sperm (monitored using 2 0 ,7 0 -bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF)), during exposure to doses of SKF or 2 mM 4aminopyridine (4-AP; timing of treatments shown with arrows). Neither 10 mM (first arrow) nor 30 mM (second arrow) SKF96365 caused a measurable increase in pHi but addition of 2 mM 4-AP (third arrow, positive control) caused an immediate rise of more than 0.5 units.

Discussion
CatSper: cation channel of sperm. occurs with SKF, its contribution to the observed elevation of [Ca 2þ ] i is likely to be small, since the effect of SKF was virtually abolished by pre-treatment with RU1968. Unlike many of these agents, the efficacy of SKF was enhanced rather than occluded by prior exposure to P4, and pre-treatment with SKF had no effect on the response to P4. This indicates that SKF and P4 activate CatSper through independent mechanisms. Since SKF did not raise pHi (indirectly activating CatSper), it appears that SKF may activate CatSper 'directly' but by a different mechanism to P4, as is the case for activation by prostaglandins (Schaefer et al., 1998;Lishko et al., 2011;Strunker et al., 2011). Future studies should address whether the action of SKF is sensitive to prior activation of CatSper by stimulation with prostaglandin.
Consistent with this interpretation of the action of SKF, assessment of CatSper activity by patch clamp showed that SKF, like P4, caused an immediate increase in current amplitude (Lishko et al., 2011;Fig. 6a). However, unlike the effect of P4, in the presence of SKF the amplitudes of both inward and outward currents then fell to levels below those observed under control conditions, even in cells where CatSper channels had previously been stimulated with P4 (Fig. 6b). This reversal of the tonic stimulation seen with P4 indicates the occurrence of a second, inhibitory action of SKF, rather than a simple decay of the stimulatory effects. SKF is not considered to be specific to CCE in its inhibitory actions, having effects on other Ca 2þ channel types including receptor-operated and voltage-operated Ca 2þ channels (VOCCs; Varnai et al., 2009). In particular, similarly to some previously characterised blockers of CatSper (e.g. Ni 2þ , NNC55-0396, Mibefradil;Lishko et al., 2011;Strunker et al., 2011), SKF has been shown to be a blocker of T-type VOCCs (Singh et al., 2010).
Dual actions of drugs (e.g. RU1968, sertraline) on [Ca 2þ ] i in human sperm, apparently reflecting an initial stimulation of CatSper, lasting a few minutes, followed by a tonic inhibitory effect, have been described previously (Rennhack et al., 2018;Rahban et al., 2021). The transient nature of the increase in CatSper current (60-100 s; Fig. 6a) is consistent with such observations and apparently provides an explanation for the similarly brief increase in [Ca 2þ ] i seen in SKF stimulated human sperm. However, other observations cannot be reconciled with this interpretation. Firstly, in cells previously exposed to P4, [Ca 2þ ] i oscillations (which require CatSper activity; Torrezan-Nitao et al., 2021) persisted for at least 10 min in the presence of SKF (often being enhanced; Fig. 1a). More strikingly, when the cells were pretreated with SKF, subsequent application of P4 (in the continued presence of SKF) was able to induce a [Ca 2þ ] i transient (known to be dependent on CatSper channels; Strunker et al., 2011) of similar amplitude to that seen in parallel control experiments (Fig. 2d). Thus, it appears that the delayed inhibition of CatSper by SKF seen in our patch clamp experiments does not occur in imaging experiments on intact cells. We are currently unable to provide an explanation for these observations. One possibility is that the blocking action of SKF is use-or voltage-dependent, such that this effect is revealed only by the repeated manipulations used in our patch clamp experiments. Future studies should address these questions by varying patch clamp protocols to test use-dependence (e.g. dependence of current decay on frequency of stimulation) and open/closed sensitivity (e.g. by varying the holding voltage of the cell during SKF pre-treatment). A further complication is that treatment with SKF96365 (5-20 mM) inhibited motility in human sperm, consistent with an inhibitory action on CatSper and/or CCE (Castellano et al., 2003), yet we observed an increase of [Ca 2þ ] i in intact cells. Regardless of the underlying mechanism, these observations suggest that patch clamp recordings of CatSper and possibly other channels in human sperm may not always reflect the behaviour of channels in intact sperm and should be interpreted cautiously.
To investigate the significance of capacitation for the action of SKF, we assessed the effects of SKF and P4 in cells where capacitation was impaired by the absence of BSA. BSA has been shown to act as an agonist of the proton channel Hv1 in human sperm and the capacitation-associated rise in pHi (and consequent increased sensitivity of CatSper to activating stimuli) is BSAdependent (Zhao et al., 2021). We found that, in the absence of BSA, both P4 and SKF were still able to induce a [Ca 2þ ] i transient in a large majority of cells, but their ability to support subsequent repetitive activity (oscillations) was markedly different. Whereas P4 induced oscillations in more than 50% of cells, SKF-generated, repetitive activity was very rare. This suggests that treatment with P4 may ameliorate the effects of BSA-free incubation, perhaps by activation of other signalling events, as has been described previously in human and primate sperm (e.g. Thomas and Meizel, 1989;Sagare-Patil et al., 2012;Sagare-Patil and Modi, 2013;Sumigama et al., 2015) leading to sensitization of CatSper. Significantly, we have also observed that stimulation with prostaglandin E 1 , which activates CatSper by a different mechanism to P4, can induce [Ca 2þ ] i oscillations both in 'capacitated' sperm and in cells prepared in the absence of BSA (Torrezan-Nitao, unpublished data). In summary, the effects of SKF on Ca 2þ signalling in human sperm are complex. The drug both stimulates and inhibits currents through CatSper channels, though the inhibitory effect is not apparent in the actions of SKF on [Ca 2þ ] i in imaged cells. These complex effects preclude the use of SKF to investigate involvement of CCE in human sperm Ca 2þ signalling.

Supplementary data
Supplementary data are available at Molecular Human Reproduction online.

Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.